Method of transporting physiological polymer using protein having rxp repeated sequence

ABSTRACT

The present invention provides a method for efficiently transporting useful protein, peptide, gene, etc. to a target cell such as a diseased cell, which is characterized by that a complex in which protein having a structure of peptide unit repetition represented by the formula Arg-Xaa-Pro (in the formula, Xaa is a hydrophobic or acidic amino acid residue) at the C-terminal of the protein is bound to at least one of useful protein, peptide, gene, etc. is administered to a living body.

TECHNICAL FIELD

The present invention relates to a method for efficiently transportingphysiological polymer such as useful protein, peptide and gene, etc. ina living body. Such a method is particularly useful in gene therapy.

BACKGROUND ART

As a result of the progress in genetic engineering in recent years,genetic abnormality that causes many diseases such as hereditary diseasehas been explicated at the DNA level, whereupon there have been inventedtherapeutic methods in which the abnormal gene is returned to normal andexpressed so that the disease is treated.

In such therapeutic methods, one of the technical challenges isdevelopment of an art for transporting useful genes efficiently andsafely to a target cell and expressing them.

With regard to a method for introducing genes into a cell,microinjection, precipitation with calcium phosphate, cation liposome,virus vector, electroporation, etc. have been usually applied. However,there are disadvantages that those methods are troublesome and also havecytotoxicity. In addition, it takes as long as 12 to 80 hours to confirmthe expression of the desired genes after introduction of genes.

In order to overcome such disadvantages, there has been developed amethod for introducing useful protein directly into a cell by usingprotein which has intercellular transportation ability. With regard tothe protein having an intercellular transportation ability, there havebeen known HIV-1 TAT (Vives, E., Brodin, P. and Lebleu, B. (1997), Atruncated HIV-1 Tat protein basic domain rapidly translocates throughthe plasma membrane and accumulates in the cell nucleus, J. Biol. Chem.272, 16010-7), antennapedia (Derossi, D., Joliot, A/H/. Chassaing, G.and Prochiantz, A. (1994), The third helix of the Antennapediahomeodomain translocates through biological membrane, J. Biol. Chem.269, 10444-50), etc. When those proteins are used, however, there is aproblem that inactivation or denaturation of the useful protein to beintroduced into a cell is resulted.

DISCLOSURE OF THE INVENTION

The present invention provides a method for binding intercellulartransportation protein to physiological polymer such as useful protein,peptide or gene and introducing the resulting complex into a target cellsuch as a diseased cell, wherein the above-mentioned problems aresolved.

The present inventors have found that useful protein, peptide or gene isable to be intercellularly transported without depending upon energysuch as ATP, when protein having repetition of tripeptide represented bythe formula Arg-Xaa-Pro (in the formula, Xaa is a hydrophobic or acidicamino acid residue) with abundant positively charged residues at theC-terminal, such as HSV US11 protein which is a US11 gene product ofHSV, interacts with negative charge of cytoplasmic membrane.

The present inventors conducted intensive investigations, and havecreated a method in which protein having a structure of peptide unitrepetition represented by the formula Arg-Xaa-Pro (in the formula, Xaais a hydrophobic or acidic amino acid residue) at the C-terminal of theprotein is bound to at least one of physiological polymer such as (a)useful protein, (b) peptide and (c) gene, etc., whereby at least one ofthe physiological polymer such as (a) useful protein, (b) peptide and(c) gene, etc. is/are introduced into a target cell such as a diseasedcell.

Thus, the present invention relates to

(1) A method for transporting at least one of (a) useful protein, (b)peptide and (c) gene in a living body, which is characterized by that atleast one of (a) useful protein, (b) peptide and (c) gene is/are boundto protein having a structure of peptide unit repetition represented bythe formula Arg-Xaa-Pro (in the formula, Xaa is a hydrophobic or acidicamino acid residue) at the C-terminal of the protein and the resultingcomplex is administered to a living body.

(2) The method mentioned in the above (1), wherein the gene is insertedinto a plasmid.

(3) The method mentioned in the above (1), wherein transport in a livingbody is intercellular transport.

(4) The method mentioned in the above (3), wherein intercellulartransport is transport to a target cell.

(5) The method mentioned in the above (4), wherein the target cell is adiseased cell.

(6) The method mentioned in the above (1), wherein the protein is US11protein of herpes simplex virus.

(7) The method mentioned in the above (1), wherein the useful protein isa cytokine or interferon-α, -β, -γ or -ω.

(8) Use of protein having a structure of peptide unit repetitionrepresented by the formula Arg-Xaa-Pro (in the formula, Xaa is ahydrophobic or acidic amino acid residue) at the C-terminal of theprotein for transporting at least one of (a) useful protein, (b) peptideand (c) gene in a living body.

(9) A transport agent for transporting at least one of (a) usefulprotein, (b) peptide and (c) gene in a living body, which comprises acomplex in which protein having a structure of peptide unit repetitionrepresented by the formula Arg-Xaa-Pro (in the formula, Xaa is ahydrophobic or acidic amino acid residue) at the C-terminal of theprotein is bound to at least one of (a) useful protein, (b) peptide and(c) gene.

(10) A complex in which protein having a structure of peptide unitrepetition represented by the formula Arg-Xaa-Pro (in the formula, Xaais a hydrophobic or acidic amino acid residue) at the C-terminal of theprotein is bound to at least one of (a) useful protein, (b) peptide and(c) gene.

(11) The complex mentioned in the above (10), wherein the gene isinserted into a plasmid.

(12) The complex mentioned in the above (10), wherein the protein isUS11 protein of herpes simplex virus.

(13) A method for the treatment of human beings or animals, which ischaracterized by that a complex in which protein having a structure ofpeptide unit repetition represented by the formula Arg-Xaa-Pro (in theformula, Xaa is a hydrophobic or acidic amino acid residue) at theC-terminal of the protein is bound to at least one of (a) usefulprotein, (b) peptide and (c) gene is administered to a patient.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, protein having repetition of tripeptiderepresented by the formula Arg-Xaa-Pro (in the formula, Xaa is ahydrophobic or acidic amino acid residue; hereinafter, it is abbreviatedas RXP) with abundant positively charged residues at the C-terminal ofthe protein is used. There is no particular limitation for Xaa so far asit is a hydrophobic or acidic amino acid residue, and examples of thehydrophobic amino acid residue are alanine, isoleucine, leucine,methionine, phenylalanine, proline, etc., while examples of the acidicamino acid residue are aspartic acid glutamic acid.

With regard to the protein used in the present invention, protein havingRXP repetition at the C-terminal may be used, and also an arbitraryprotein where RXP repetition is introduced into the C-terminal may beused. Introduction of RXP repetition into the C-terminal may be carriedout by a known method per se. The numbers of RXP repetition at theC-terminal may be about 10 to 40.

A preferred example of the protein having RXP repetition at theC-terminal is HSV US11 protein which is a US11 gene product of herpessimplex virus (hereinafter, abbreviated as HSV). The number of RXPrepetition at the C-terminal of HSV US11 is 19.

The method for the production of pure HSV US11 protein is not limited tothe following method and may be carried out according to known methods.A preferred method for the production of pure HSV US11 protein is shownbelow.

An open reading frame (ORF) of HSV US11 is located between the positions147247 and 147699 of a nucleotide of HSV-2 genome. A base sequencecoding for HSV US11 protein is amplified from HSV-2186 genomic DNA bypolymerase chain reaction (hereinafter, abbreviated as PCR method),wherein US11-F (ATTAGGATCCATGGCATCCGGGGTTTCC) and US11-R(TTATGCGGCCGCCTAGGCAAGCCCGCGGGT) are used as a forward primer and areverse primer, respectively.

The resulting PCR product is cleaved by Sal I and Not I and insertedinto a frame of Escherichia coli expression vector pET-28a (manufacturedby Novagen) to prepare a plasmid pET-28a-US11 followed by being cloned.Lac operator and IPTG-inducible tac promoter are used to control theexpression. The plasmid is transformed into Escherichia coli BL21DE3strain (manufactured by Novagen) to express a large quantity of6×His-tagged US11 fusion protein. The 6×His-tagged US11 fusion proteinexpression cells are dissolved and separated in PBS using ultrasonicwave, the cell pieces are removed by centrifugation, and the6×His-tagged US11 fusion protein is eluted with a fraction containing300 mM imidazole, thus pure HSV US11 protein is obtained.

There is no particular limitation for useful protein which may be usedin the present invention, and protein which is useful for the preventionor treatment of diseases of human beings or animals is preferred. Apharmaceutically effective, useful protein which has been difficult tobe incorporated into cells such as diseased cells is particularlypreferred. Any protein may be used as useful protein, and its examplesare a cytokine, interferon-α, β, γ or ω, interferon of vertebrates, andanimal-derived interferon such as canine interferon (α, β and γ type)and feline interferon ω. Other examples are hemagglutinin(hemagglutinative agent) of influenza A or B virus, HA 2 component ofhemagglutinin of influenza A or B virus and peptide analogs thereof, M2protein of influenza A virus and HEF protein of influenza C virus.

With regard to the useful protein, pure one can be procured according toa known method. Useful protein can be prepared by such a manner that,taking its properties such as stability to salt, hydrophobicity andbiological specificity into consideration, impurities such as proteaseare removed by various types of chromatography, and the aimed protein isrecovered and concentrated. Examples of the chromatography are gelfiltration chromatography, ion-exchange chromatography, reversed phasechromatography, affinity chromatography and hydrophobic chromatography.

With regard to the peptide which may be used in the present invention,any peptide may be used so far as it is a peptide useful in a livingbody, and such peptide is obtained, for example, by modifying theabove-mentioned useful protein into peptide form, and peptide stillmaintaining the utility of the useful protein is preferred. There ispreferably listed a peptide where protein useful for the prevention ortreatment of diseases of human beings or animals is modified. Withregard to a means for producing peptide by modification of protein, amethod for decomposition of protein such as hydrolysis may beexemplified. It may also be a peptide prepared by mixing individualamino acids. Although a peptide is usually produced by introducing aminoacids step by step according to an organic chemical synthetic method, itmay also be produced by other methods such as enzymatic hydrolysis ofnatural protein or peptide synthesis by an enzymatic method.

With regard to a method of introducing amino acids step by stepaccording to an organic chemical synthetic method, solid phase peptidesynthesis or liquid phase peptide synthesis has been known, and they arementioned in detail, for example, in Fundamentals and Experiments ofPeptide Syntheses by Nobuo Izumiya, et al. (Maruzen).

With regard to a method for producing peptide by enzymatic hydrolysis ofnatural protein, for example, a method where milk casein is hydrolyzedwith trypsin or a method where corn protein is hydrolyzed withmicroorganism-derived thermolysin to prepare hypotensive peptide hasbeen known (Susumu Maruyama, Bioscience and Industry, volume 47, pages38 to 42, 1989).

With regard to a method for peptide synthesis by means of an enzymaticmethod, there are two methods—condensation and substitution—and in theformer, an amine component is substituted for a carboxyl componenthaving a free carboxyl group, and in the latter, an amine component issubstituted for an esterified or amidated carboxyl component. All ofthem may be carried out according to a known method.

The present invention can be used for introducing gene which is usefulfor a living body into a living body. For example, gene which is usefulfor the prevention and treatment of diseases of human beings or animalsor, in other words, gene which is used in gene therapy is listed. It ispreferred that the gene is in a form of a plasmid. The gene to beinserted into a vector may be prepared, for example, by conducting PCRutilizing a primer DNA, which is designed and prepared on the basis of abase sequence of the original protein using, as a template, a genomicDNA of a living body (cDNA, when the living body is an eukaryote) whichproduces useful protein (hereinafter, referred to as original protein)expressing in a cell. cDNA can be directly amplified by means of areverse transcriptase polymerase chain reaction (hereinafter,abbreviated as RT-PCR method) using total RNA or an mRNA fraction whichis prepared from cells/tissues. Also, those produced according to asynthetic method for an oligonucleotide known per se may be used. To bemore specific, a chemical synthesis method using a DNA synthesizer suchas a DNA synthesizer model 392 (manufactured by Perkin-Elmer) utilizinga phosphoamidite method may be listed.

With regard to a method for the preparation of genomic DNA or cDNA ofthe living body which produces the original protein, it is appropriatelyselected depending upon the type of the living body and is notparticularly limited. For example, it is possible to apply a methodmentioned in Molecular Cloning, 2nd Ed., Cold Spring Harbor LaboratoryPress (1989), J. Sambrook, et al.

With regard to gene to be introduced into a target cell (such as adiseased cell), there is no particular limitation and, for example, genefor the diseases, i.e. gene which antagonistically acts on the disease,gene which supplements the deficiency in the disease, etc. may be used.With regard to the genes as such, gene which expresses theabove-mentioned useful protein in a living body may be specificallylisted, and its example is gene which codes for a pharmacologicallyactive substance selected from the group consisting of cytokine, growthfactor, antibody or antibody fragment, receptor to cytokine or growthfactor, protein having effect of growth inhibition or cell growthsuppression, enzyme, thrombus-inducing substance,agglutination-inhibiting substance, protein having fibrinolysis action,virus coat protein, bacterial antigen and parasitic antigen, tumorantigen, protein effecting on blood circulation, peptide hormone andribonucleic acid such as ribozyme and antisense RNA.

Further, examples of representative gene to specific diseases are asfollows. To inflammatory diseases, they are genes coding for SOD,anti-inflammatory cytokines and peptide antagonistically acting on celladhesion factors; to enzyme deficiency, they are genes coding for normalenzymes; to receptor deficiency, they are genes coding for normalreceptors; to viral infection, they are genes coding for thymidinekinase for killing/damaging virally infected cells, genes coding for atoxin such as diphtheria toxin, etc., and genes coding for antisense,triple helix, ribozyme, decoy, transdominant mutant, etc. which inhibitthe duplication of virus; to cancer, they are genes coding for thymidinekinase for killing/damaging cancer cells, genes coding for a toxin suchas diphtheria toxin, etc., genes coding for antisense, ribozyme, triplehelix, etc. for inactivating the cancer genes, genes coding forcancer-suppressing genes such as p53 for normalizing the cancer cells,and genes coding for antisense, triple helix, ribozyme, etc. forinactivating the genes participating in multidrug resistance toanti-cancer agents; to familial hypercholesterolemia, they are genescoding for LDL receptors; etc.

The gene to be used in the present invention may be prepared by beinginserted into a duplicable plasmid vector, a phage vector, etc. in atarget cell according to a common method. In the present invention, aform in a plasmid is preferred. With regard to a vector, it is preferredto use an expression vector which is prepared in such a manner thatstable mRNA is able to be generated in large quantities and theresulting mRNA is efficiently translated also in a target cell. Whenplural genes are introduced, it is preferred to use vectors havingdifferent origins of replication (ori) even if they are same genes ordifferent genes.

With regard to a plasmid vector, it may have drug-resistance genes suchas erythromycin-resisting gene, neomycin-resisting gene,kanamycin-resisting gene, etc. in view of being able to easily screen atransformant having a recombined vector to be prepared and also beingable to stably hold the recombined vector in the target cell.

With regard to a plasmid vector used for insertion of genes, examplesthereof include plasmid pET-28a, pFLAG-CMV-5a, pCDM8, pcDNAI/Amp,pcDL-SRα, BCMGSNeo, pSV2-neo, pSV-2gpt, pEF-BOS, pCEV4, pME 18S, pKY4,pKK223-3, pVL 1392, pVL 1393, Ti plasmid, Ri plasmid, pBI 121, pUB 110,pUC 118, etc. The plasmid may be appropriately selected depending uponthe type of the gene to be inserted, and commercially available plasmidsmay be used. Examples of the commercially available plasmids are pET-28a(manufactured by Novagen) and pFLAG-CMV-5a (manufactured by Novagen),etc.

With regard to a phage vector, λgt 11 phage, λgt 10 phage, etc. may beutilized. Any of them gives a recombinant vector corresponding to theparent vector used. The phage vector may be appropriately selecteddepending upon the gene to be inserted, and a commercially available onemay also be used.

General means for the preparation of recombinant plasmids is describedin Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press,(1989) J. Sambrook, et al.

With regard to an expression cassette used for the gene to be inserted,anything may be used without particular limitation so far as it is ableto express the gene in a target cell. As for the expression cassette, anexpression cassette which is able to express the gene in animal-derivedcells may be used. Preferably, it is an expression cassette which isable to express the gene in mammalian-derived cells and, morepreferably, it is an expression cassette which is able to express thegene in human-derived cells.

The expression vector usually contains a regulatory sequence to expressthe gene or to be advantageous for the expression. Each regulatorysequence may be native or foreign to a base sequence coding for an aminoacid sequence of the gene. Such a regulatory sequence includes promoter,leader, polyadenylated sequence, propeptide sequence, enhancer, signalsequence, splicing signal, poly A-added signal, SV 40 duplicated originand transcription terminator, although they are not limitative. Amongthem, it is preferred that the regulatory sequence includes at leastpromoter and transcription- and translation-termination signals.

With regard to a promoter, anything may be used so far as it is anappropriate promoter sequence which is a base sequence recognizable inthe target cell. Examples thereof are lac promoter, SRα promoter, SV 40promoter, LTR promoter, CMV (cytomegalovirus) promoter and HSV-TKpromoter, etc.

With regard to the transcription terminator, anything may be used so faras it is a sequence which is recognizable in a target cell forcompleting the transcription and, for example, transcription terminatorsequences of genes derived from virus, various mammals and birds may beused. To be more specific, SV 40 terminator of simian virus, etc. may beused.

The expression vector also includes a signal sequence concerningsecretion of protein. As for the signal sequence, either a signalsequence of the gene to be introduced or a signal sequence of differentgene may be used. For example, insulin signal sequence, α-interferonsignal sequence, antibody signal sequence, etc. may be used.

The expression vector may include one or more factor(s) advantageous forexpression of enzyme gene such as one or more nucleic acid sequence(s)coding for activator (such as trans-acting factor), chaperone andprocessing protease. Any factor which is functional in a target cell mayalso be used as an expression vector according to the present invention.

Protein having RXP repetition at the C-terminal forms a complex withuseful protein, peptide, a plasmid into which gene is inserted, etc. bya non-covalent bond. The complex of protein having RXP repetition at theC-terminal with useful protein, peptide, a plasmid into which gene isinserted, etc. moves into nucleolus of the target cell and itssurrounding cells.

In the case when a complex is formed with useful protein having amolecular weight of more than 10 kDa, protein having RXP repetition atthe C-terminal is added to, for example, PBS or serum-deficient mediumin an aseptic tube preferably in an amount of about 1/100 (w/w) relativeto the total amount. After that, about 0.25 to 1 part by weight of theuseful protein is added thereto, followed by mixing.

In the case of a plasmid into which peptide, gene or a low-molecularuseful protein of not more than 10 kDa is inserted, a protein having RXPrepetition at the C-terminal which is diluted to about 1/10 (w/w) withPBS is added to PBS or serum-deficient medium in an aseptic tube in anamount of about 1/100 (w/w) relative to the total amount. After that,about 100 to 500 parts by weight of peptide or low-molecular usefulprotein of not more than 10 kDa is added thereto, followed by mixing.

After mixing, the mixture is incubated at about 15 to 30° C. or,preferably, at about 20 to 25° C. for about 20 to 50 minutes orpreferably about 30 to 40 minutes to form a complex. The complexprepared as such is administered to a living body. With regard to amethod for the administration to a living body, a method where it isdirectly administered to a living body by means of injection or thelike, a method where a target cell such as a diseased cell is extractedfrom the body of a patient, transformed and returned to a living body,etc. may be exemplified and any of the methods may be used.

With regard to a method for direct administration to a living body,there is no particular limitation and, for example, it is possible totopically administer into vein, artery, portal vein, cranium, pleura,peritoneum, etc. by means of injection, etc.

Since the dose varies depending upon the type, etc. of protein havingRXP repetition at the C-terminal, useful protein, peptide or a plasmidinto which gene is inserted, it cannot be determined unconditionally. Inthe case of adults, however, it is usually preferred to administer 1 to1,000 mg/kg of a complex. The dose may be appropriately determined by amedical doctor taking the patient's state, condition of the disease,etc. into consideration.

With regard to a method where the target cells are extracted from thebody of a patient, transformed and then returned into the body again,firstly the target cells are extracted from the body of a patient. Thereis no particular limitation for such cells, and examples thereof includebone marrow cell, myeloma cell, liver cell, alveolus cell, muscle cell,lymph cell, lymphoadenoma cell, mammotropic cell, dermatofibroblast,epithelial cell, endothelial cell, interstitial cell, parenchymal cellof organ such as liver, kidney, spleen, thymus, pancreas, placenta,uterus and lung, cartilage cell, nerve cell and the like. With regard toa method for extracting the cells from the body of a patient, there is amethod where, for example, body fluid such as blood is collected fromthe patient, although there is no particular limitation and that may becarried out according to a known method.

Further, the present invention may also be used for various mammaliancells such as HS-68 cell, NIH 3T3 cell, C2C12 cell, CEM-SS cell, HEPGcell, Hela cell, monkey COS-7 cell, Vero cell, Chinese hamster cell CHO(hereinafter, abbreviated as CHO cell), dhfr gene-deficient Chinesehamster cell CHO (hereinafter, abbreviated as CHO(dhfr⁻) cell), mouse Lcell, mouse AtT-20 cell, mouse myeloma cell, rat GH3 cell, human FLcell, 293 cell, C127 cell, BALB/3T3 cell, Sp-2 cell, etc.

The above-mentioned cells are proliferated on a medium under a conditionfor accelerating the proliferation. With regard to the medium, a mediumwhich contains carbon source and nitrogen source may be used. Withregard to the carbon source, sugar, organic acid, etc. may beexemplified, and examples of the sugar are glucose, glycerol, starch,dextran, molasses, etc. With regard to the nitrogen source, organicnitrogen source, inorganic nitrogen source, etc. may be exemplified, andexamples of the organic nitrogen source are casein, peptone, meatextract, yeast extract, Casamino acid, glycine, etc. and examples of theinorganic nitrogen source are ammonium sulfate, ammonium nitrate, etc.

In order for the transformation to take place, the medium is removedfrom the cells and the cells are washed with PBS, and then the complexprepared by the above-mentioned condition is added to the desired cellsand incubated in a moisturized air containing about 5% of CO₂ for about1 hour or longer at about 35 to 40° C. or, preferably, about 37° C. sothat the above complex is well contacted to the cells.

After that, about 1 ml of the completely grown medium is added to thecells and incubated at about 15 to 42° C., or preferably about 30 to 40°C. for about 0.5 to 2 hour(s) or, preferably, for about 0.5 to 1 hour inthe case of useful protein or for about 1 to 2 hour(s) in the case ofpeptide, low-molecular useful protein or a plasmid. In that case,aeration or stirring may be carried out if desired.

If necessary, a nutrient matter may be added to the medium. Examples ofsuch nutrient matter are serum, amino acid, vitamin, nucleic acid, salt,etc. With regard to the medium, a synthetic medium mainly comprisingsugar and inorganic nitrogen source, for example, may be used. When asolid medium is used as a medium, agar is further added thereto.

For the purpose of keeping the transformant stably and suppressing thegrowth of the strain having no transformant, an antibiotic substance maybe added to the medium. Examples of such antibiotic substance arepenicillin, erythromycin, neomycin, chloramphenicol, bacitracin,D-cycloserine and ampicillin. Further, anti-foaming agent such assoybean oil, lard oil and various surface-active agents, etc. may beadded to the medium if necessary. The pH of the medium is usually about5 to 9 in view of the pH where the transformant is able to grow, andpreferably it is about 6.5 to 7.5.

The resulting transformant is returned to the body of the patient. Thereis no particular limitation for a method for the process, and it ispossible to administer to a patient who needs the treatment by, forexample, intravenous administration, topical administration to thediseased part, oral administration, etc. If it is necessary for thepurpose of the administration, a pharmaceutically acceptable additivesuch as carrier, excipient, stabilizer, dissolving aid, or etc. is addedthereto to prepare a pharmaceutical preparation suitable for theabove-mentioned administration. Incidentally, general procedures forconducting such genetic therapies are fully described, for example, in“Special Issue of Jikken Igaku; Biomanual UP Series—FundamentalTechnique of Gene Therapy” edited by Takashi Shimada, Izumi Saito andToshiya Ozawa (1996), Yodosha.

EXAMPLES Test Example 1

Vero cells which are stable strain of renal cells of African greenmonkey were grown in an Eagle's minimum essential medium (MEM)containing 5% bovine serum.

In order to check the intercellular transportation ability of HSV-2protein, plasmid pcDNA-US11 was constructed. PCR primers were US11Bam HI(ATTAGGATCCATGGCATCCGGGGTTTCC) and US11-R. A PCR product was cleaved byBam HI and Not I and then cloned by pcDNA 3.1(+). Transfection into thevero cells was conducted according to a protocol recommended by GibcoBRL using a lipofectamine reagent.

Transfection of pcDNA-US11 into the cells was investigated by anindirect immunofluorescence technique using antiserum. The antiserum wasproduced by conducting immunoreactions by MPL+TDM+CWS emulsion adjuvantsystem (RIBI ImmunoChem Research, Inc., Montana) using two rabbits andusing an emulsion containing about 0.6 mg of HSV US11 protein to which6×His expressed in Escherichia coli was added. Inoculation was carriedout by a hypodermic injection into a shaved back. The same adjuvant andpure protein (0.6 mg) were used for continuous stimulation. Stimulationwas applied for three times in total, each stimulation being appliedwith an interval of two weeks after the first injection. After two weeksfrom the final immunoreaction, blood was collected from the heart.Rabbit polyclonal anti-US11 antiserum strongly reacts with proteins ofapparent molecular weights of 20 kDa and 21 kDa existing in dissolvedHSV-2 infected cells.

The cells were washed with PBS and fixed with 4% paraformaldehyde (PFA)at 4° C. for 45 minutes. After fixed with PFA, they were washed for 45minutes with PBS containing 0.05% Triton X-100.

Rabbit polyclonal anti-US11 antiserum was diluted to an extent of 1/500(w/w) and was used as a primary antibody. After incubating at 37° C. for30 minutes, a cover glass was washed in PBS and labeled with thesecondary antibody at 37° C. for 30 minutes.

FITC-conjugated goat anti-rabbit IgG (manufactured by MBL) was used as asecondary antibody. After washing with PBS, the cover glass was quicklyplaced on a slide glass using PermaFluor (manufactured by Immunon).After that, analysis was conducted by a ZEISS laser scanning microscopeLSM 510.

Fluorescence of US11 was observed not only in nucleoluses but also incytoplasms. Moreover, strong fluorescence was also detected innucleoluses of many cells around them.

Test Example 2

In order to further investigate the spread of HSV US11 protein among thecells, pFLAG-US11 which expresses US11-FLAG fusion protein wasconstructed and its activity was investigated. In that process, ananti-FLAG mouse monoclonal antibody was used as the primary antibody forthe detection of US11-FLAG fusion protein.

In order to prepare pFLAG-US11 which expresses US11-FLAG fusion protein,PCR products of US11GFPFW and US11GFPRv− were introduced into Bgl II/SalI site of pFLAG-CMV-5a to prepare pFLAG-US11, followed by cloning.Plasmid pFLAG-CMV-5a (Sigma) expresses US111-FLAG fusion protein underthe control of HCMV immediate early promoter.

Indirect immunofluorescence measurement was carried out by the samemanner as in Test Example 1, except that an anti-FLAG mouse monoclonalantibody (manufactured by Sigma) diluted to an extent of 1/100 (w/w) wasused as the primary antibody and FITC-conjugated goat anti-mouse IgG(manufactured by Sigma) was used as the secondary antibody. Theanti-FLAG mouse monoclonal antibody recognizes a base sequencecomprising eight amino acids (Asp-Try-Lys-Asp-Asp-Asp-Asp-Lys)positioned at KpnI site and thereafter in MCS of a pFLAG-CMV-5a vector.

Both nucleoluses and cytoplasms were strongly stained in small numbersof cells, and further in the cells surrounding them, nucleoluses werestained. Therefore, most of cells showed a nucleolus-staining pattern.

Test Example 3

In order to investigate whether an intercellular transportation abilityof HSV US11 protein is maintained when bigger polypeptide is fused,US11-EGFP or EGFP-US11 expression plasmid was constructed.

In order to express a fusion protein of US11 with EGFP, pEGFP-C1-US11and pEGFP-N-3-US11 were constructed. For both of them, the forward PCRprimer was US11GFPFW (AATCAGATCTATGGCATCCGGGGTTTCC) in which Bgl II sitewas integrated. The reverse primer to pEGFP-C1-US11 wasUS11GFPRv+(AATCGTCGACCTAGGCAAGCCCGCGGGT) and the reverse primer topEGFP-N-3-US11 was US11GFPRv−(AATCGTCGACGGCAAGCCCGCGGGTTGC) integratedin a Sal I site.

A PCR product of each pair was cleaved with Bgl II/Sal I as a Bgl II-SalI fragment and inserted into pEGFP-C1 and pEFGP-N3 to preparepEGFP-C1-US11 and pEGFP-N-3-US11.

A plasmid was transfected to a cell in the same way as Test Example 1,and 40 hours later, the cell was fixed with cold acetone. Indirectimmunofluorescence measurement was carried out by the same manner as inTest Example 1 using a rabbit polyclonal anti-US11 antiserum as aprimary antibody and an FITC-conjugated goat anti-rabbit IgG(manufactured by MBL) as a secondary antibody.

In any of US11-EGFP or EGFP-US11, fluorescence was observed in bothnucleoluses and cytoplasms in the transfected cells and also in cellssurrounding them. Moreover, most of the cells showed a nucleolus-stainedpattern. Those result show that intercellular transfection activityamong the cells is maintained in those fusion proteins.

Test Example 4

Investigation was carried out to examine whether extrinsic HSV US11protein was transfected into cells.

US11 fusion protein to which pure 6×His was added was dialyzed againstPBS containing 100 mM NaCl and 10% glycerol and diluted with OPTI MEM(manufactured by Gibco BRL). Vero cells which were a stable strain ofrenal cells of African green monkey grown in an Eagle's minimumessential medium (MEM) containing 5% bovine serum were washed with PBSfor two times. After that, the cells were incubated at 37° C. for 10minutes in the presence of various concentrations of pure HSV US11protein. After well washed with cold PBS once again, the cells werefixed with cold acetone, and indirect immunofluorescence measurement wasconducted by the same manner as in Test Example 1 using a rabbitpolyclonal anti-US11 antiserum as a primary antibody and anFITC-conjugated goat anti-rabbit IgG antibody as a secondary antibody.

Although the US11-specific fluorescence was mostly observed innucleoluses, it was also able to be detected not only in nucleoluses butalso in cytoplasms when incubation was conducted in HSV US11 proteinhaving a higher concentration. Those results show that extrinsic HSVUS11 protein moves into cells.

Test Example 5

In order to investigate whether an energy-dependent route such asendocytosis participates in internalization of HSV US11 protein incells, the amount of cells ingested by US11 fusion protein to which6×His was added was tested under the condition of 4° C. by the samemanner as in Test Example 4.

The 6×His-added US11 fusion protein moves into the cells at 4° C. andwas found to be accumulated mostly in nucleoluses. Fluorescence ofcytoplasms was stronger in the cells incubated at 4° C. than thoseincubated at 37° C. Therefore, it was noted that internalization of theHSV US11 protein in cells was in accordance with an energy-independentroute.

Example 1

A protein which induces the production of interferon-γ (hereinafter,abbreviated as IFN-γ) in immune competent cells was used as usefulprotein. An HSV US11 protein of herpes simplex virus was used as aprotein having RXP repetition at the C-terminal.

Pure IFN-γ-producing protein was prepared according to the followingmethod. Dead cells prepared by heating Corynebacterium parvum (ATCC11827) at 60° C. for 1 hour were administered by injection intoperitoneums of 600 female CD-1 mice of 8 weeks age in an amount of 1 mgper a mouse and, after breeding the mice for seven days by a common andgeneral method, pure lipopolysaccharide derived from Escherichia coliwas injected into vein in an amount of 1 μg per a mouse. After 1 to 2hour(s), mice were killed by dislocation of cervical vertebra, blood wascollected from the heart, and then the liver was excised, crushed by ahomogenizer in 8-fold volume of 50 mM phosphate buffer (pH 7.3) andextracted. The extract was centrifuged at about 8,000 rpm for 20minutes, 50 mM phosphate buffer (pH 7.3) containing saturated ammoniumsulfate was added to about 9 liters of the resulting supernatant liquidso as to make ammonium sulfate 45% saturation, and the mixture wasallowed to stand at 4° C. for 18 hours and centrifuged at about 8,000rpm for 30 minutes to give about 19 liters of supernatant liquidcontaining the protein.

The supernatant liquid was loaded on 4.6 liters of a column ofphenyl-Sepharose (manufactured by Pharmacia) which was previouslyequilibrated with 50 mM phosphate buffer (pH 7.3) containing 1M ammoniumsulfate and, after the column was washed with the fresh same buffer, 50mM phosphate buffer (pH 7.3) was passed through the column at SV 0.57under a concentration gradient of ammonium sulfate descending from 1M to0.2M. A fraction (4.8 liters) containing the protein which eluted whenthe ammonium sulfate concentration was about 0.8 M was collected,subjected to membrane concentration, dialyzed against 20 mM phosphatebuffer (pH 6.5) at 4° C. for 18 hours, then loaded on a column of 250 mLof DEAE-Sepharose (manufactured by Pharmacia) which was previouslyequilibrated with 20 mM phosphate buffer (pH 6.5). The column was washedwith the fresh same buffer, and then 20 mM phosphate buffer (pH 6.5) waspassed through the column at SV 1.2 under a concentration gradient ofsodium chloride ascending from 0 M to 0.2 M, whereupon the proteineluted when sodium chloride concentration was about 0.13 M.

The eluate (260 mL) containing the protein was collected, concentrated,dialyzed against 25 mM bis-Tris buffer (pH 7.1) at 4° C. for 18 hoursand loaded on 24 mL of column of Mono-P (manufactured by Pharmacia)which was previously equilibrated with the fresh same buffer, and 10%(v/v) Polybuffer 74 (pH 4.0) was passed through the column under a pHgradient descending from pH 7 to pH 4, whereupon the protein eluted whenpH was 4.8. The eluate (23 mL) containing the protein was collected,concentrated, loaded on a column of Superdex 75 (manufactured byPharmacia) which was previously equilibrated with a mixed solution (pH7.2) of 7 mM disodium hydrogen phosphate, 3 mM sodium dihydrogenphosphate and 139 mM sodium chloride, and subjected to a gel filtrationchromatography by passing the fresh same mixed solution therethrough,whereupon the protein eluted where the molecular weight was about 19,000Dalton. A fraction containing the protein was collected. The yield wasabout 0.6 μg per a mouse.

Pure HSV US11 protein was prepared by the following method. Firstly, abase sequence coding for HSV US11 protein was amplified from HSV-2186genomic DNA by polymerase chain reaction (hereinafter, referred to asPCR) using US11-F (ATTAGGATCCATGGCATCCGGGGTTTCC) as a forward primer andUS11-R (TTATGCGGCCGCCTAGGCAAGCCCGCGGGT) as a reverse primer.

The resulting PCR product was cleaved with Sal I and Not I and insertedinto a frame of Escherichia coli expression vector pET-28a (manufacturedby Novagen), whereupon a plasmid pET-28a-US11 was prepared and then itwas cloned. The plasmid was transformed into Escherichia coli BL21DE3strain (manufactured by Novagen), and 6×His-added US11 fusion proteinwas expressed in large quantities. The 6×His-added US11 fusion proteinexpression cells were dissolved and separated by ultrasonic wave in PBS,cell pieces were removed by centrifugation, and the 6×His-added US11fusion protein was obtained by eluting with a 300 mMimidazole-containing fraction.

PBS (5 mL) was added to a sterilized tube, and then 50 μL of the HSVUS11 protein prepared by the above method and 0.5 μg of IFN-γ-producingprotein were added thereto, incubated at 25° C. for 30 minutes to give acomplex of HSV US11 protein with IFN-γ-producing protein.

INDUSTRIAL APPLICABILITY

According to the present invention, useful protein, peptide, gene, etc.are efficiently transported to cells in a living body. In addition,according to the present invention, when the useful protein istransported to the cells, it is possible to transport it without causinginactivation or denaturation of the useful protein, and further, even adrug which is difficult to put into cells is able to be easily anddirectly introduced into the cells, whereby a significant effect of thedrug can be achieved.

1. A method for transporting at least one of (a) useful protein, (b)peptide and (c) gene in a living body, which is characterized by that atleast one of (a) useful protein, (b) peptide and (c) gene is/are boundto protein having a structure of peptide unit repetition represented bythe formula Arg-Xaa-Pro (in the formula, Xaa is a hydrophobic or acidicamino acid residue) at the C-terminal of the protein and the resultingcomplex is administered to a living body.
 2. The method according toclaim 1, wherein the gene is inserted into a plasmid.
 3. The methodaccording to claim 1, wherein transport in a living body isintercellular transport.
 4. The method according to claim 3, whereinintercellular transport is transport to a target cell.
 5. The methodaccording to claim 4, wherein the target cell is a diseased cell.
 6. Themethod according to claim 1, wherein the protein is a US11 protein ofherpes simplex virus.
 7. The method according to claim 1, wherein theuseful protein is a cytokine or interferon-α, -β, -γ or -ω.
 8. Use of aprotein having a structure of peptide unit repetition represented by theformula Arg-Xaa-Pro (in the formula, Xaa is a hydrophobic or acidicamino acid residue) at the C-terminal of the protein for transporting atleast one of (a) useful protein, (b) peptide and (c) gene in a livingbody.
 9. A transport agent for transporting at least one of (a) usefulprotein, (b) peptide and (c) gene in a living body, which comprises acomplex in which protein having a structure of peptide unit repetitionrepresented by the formula Arg-Xaa-Pro (in the formula, Xaa is ahydrophobic or acidic amino acid residue) at the C-terminal of theprotein is bound to at least one of (a) useful protein, (b) peptide and(c) gene.
 10. A complex in which protein having a structure of peptideunit repetition represented by the formula Arg-Xaa-Pro (in the formula,Xaa is a hydrophobic or acidic amino acid residue) at the C-terminal ofthe protein is bound to at least one of (a) useful protein, (b) peptideand (c) gene.
 11. The complex according to claim 10, wherein the gene isinserted into a plasmid.
 12. The complex according to claim 10, whereinthe protein is a US11 protein of herpes simplex virus.
 13. A method forthe treatment of human beings or animals, which is characterized by thata complex in which protein having a structure of peptide unit repetitionrepresented by the formula Arg-Xaa-Pro (in the formula, Xaa is ahydrophobic or acidic amino acid residue) at the C-terminal of theprotein is bound to at least one of (a) useful protein, (b) peptide and(c) gene is administered to a patient.